The overall goal of the proposed research is to determine the three-dimensional structures of the FXYD family of small, homologous, ion transport regulators or channels, in phospholipid bilayer membranes, using NMR spectroscopy. The proteins of this family are implicated in various diseases, including breast cancer and heart disease, and include the Mammary tumor (Mat8) membrane protein which is expressed both in primary human breast tumors and breast tumor cell lines, and is believed to be a marker of specific oncogenic cell transformation. Mat8, like the other members of the FXYD family, functions as an ion channel or channel modulator, involved in the regulation of transmembrane ion flux and transport across breast epithelial tissue. The ion channel activities of Mat8, and of the other FXYD proteins, provide the initial insight into their mechanisms of action and potential as a drug receptors: since inhibitors of the Mat8 channel may kill breast cancer cells, this protein is an attractive candidate for the development of anti-cancer drugs. The structure of Mat8 is the starting point for the rational screening and design of molecules that interfere with its function. The structures of the FXYD membrane proteins will be determined using solid state and solution NMR spectroscopy of isotopically labeled samples of the proteins in lipid bilayers or in lipid micelles. The procedure for structure determination by NMR spectroscopy involves the following steps: (1) the expression and purification of milligram quantities of isotopically labeled recombinant proteins; (2) the preparation of samples of isotopically labeled protein in oriented lipid bilayers for solid state NMR spectroscopy, and in isotropic lipid micelles for solution NMR spectroscopy; (3) the execution of multi-dimensional solid state and solution NMR experiments needed for structure determination; and (4) the analysis of experimental data to determine the protein structure in the membrane. An important goal of this research will be to correlate the structures with the ion channel activities of the FXYD proteins, determined through concentrative ion uptake assays of the proteins reconstituted in liposomes, and by measuring channel recordings of the proteins reconstituted in planar lipid bilayers. The three-dimensional structures of the FXYD proteins, correlated with their ion channel activities, are an important step towards understanding their functions. They are also the starting point for the development of inhibitor molecules targeted to breast cancer and other diseases.